Yang Hailian, Barros-Rios Jaime, Kourteva Galina, Rao Xiaolan, Chen Fang, Shen Hui, Liu Chenggang, Podstolski Andrzej, Belanger Faith, Havkin-Frenkel Daphna, Dixon Richard A
BioDiscovery Institute, University of North Texas, Denton, TX 76203, USA.
BioDiscovery Institute, University of North Texas, Denton, TX 76203, USA; Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA.
Phytochemistry. 2017 Jul;139:33-46. doi: 10.1016/j.phytochem.2017.04.003. Epub 2017 Apr 12.
A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a "vanillin synthase" could enable biotechnological production of vanillin from ferulic acid using a "natural" vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined.
最近的一篇出版物描述了一种来自香草兰（Vanilla planifolia）的酶，它能够将阿魏酸直接转化为香草醛。作者提出，这代表了香草醛生物合成的最后一步，随后香草醛通过糖基化作用转化为其储存形式——葡萄糖香草醛。这种“香草醛合酶”的存在可能使利用“天然”香草酶从阿魏酸生物技术生产香草醛成为可能。所提出的香草醛合酶与半胱氨酸蛋白酶具有高度同源性，并且在蛋白质序列水平上与2003年鉴定出的一种与将4-香豆酸转化为4-羟基苯甲醛相关的蛋白质相同。我们在此证明，无论是在体外转录-翻译系统、大肠杆菌、酵母还是植物中表达的重组类半胱氨酸蛋白酶蛋白，都无法将阿魏酸转化为香草醛。相反，该蛋白是一种酶复合物的组成部分，该复合物优先将4-香豆酸转化为4-羟基苯甲醛，对该复合物的纯化和肽测序证明了这一点。此外，RNA测序提供了证据表明该蛋白在香草兰的许多组织中都有表达，无论这些组织是否产生香草醛。基于我们的结果，香草兰似乎不含有一种能够自身直接将阿魏酸转化为香草醛的类半胱氨酸蛋白酶“香草醛合酶”。香草兰中香草醛的合成途径仍有待最终确定。
